title: Simplifying the synthesis of SIgA: combination of dIgA and rhSC using affinity chromatography.
abstract: The mucosal epithelia together with adaptive immune responses, such as local production and secretion of dimeric and polymeric immunoglobulin A (IgA), are a crucial part of the first line of defense against invading pathogens. IgA is primarily secreted as SIgA and plays multiple roles in mucosal defense. The study of SIgA-mediated protection is an important area of research in mucosal immunity but an easy, fast and reproducible method to generate pathogen-specific SIgA in vitro has not been available. We report here a new method to produce SIgA by co-purification of dimeric IgA, containing J chain, and recombinant human SC expressed in CHO cells. We previously reported the generation, production and characterization of the human recombinant monoclonal antibody IgA2 b12. This antibody, derived from the variable regions of the neutralizing anti-HIV-1 mAb IgG1 b12, blocked viral attachment and uptake by epithelial cells in vitro. We used a cloned CHO cell line that expresses monomeric, dimeric and polymeric species of IgA2 b12 for large-scale production of dIgA2 b12. Subsequently, we generated a CHO cell line to express recombinant human secretory component (rhSC). Here, we combined dIgA2 b12 and CHO-expressed rhSC via column chromatography to produce SIgA2 b12 that remains fully intact upon elution with 0.1M citric acid, pH 3.0. We have performed biochemical analysis of the synthesized SIgA to confirm the species is of the expected size and retains the functional properties previously described for IgA2 b12. We show that SIgA2 b12 binds to the HIV-1 gp120 glycoprotein with similar apparent affinity to that of monomeric and dimeric forms of IgA2 b12 and neutralizes HIV-1 isolates with similar potency. An average yield of 6 mg of SIgA2 b12 was achieved from the combination of 20mg of purified dIgA2 b12 and 2L of rhSC-containing CHO cell supernatant. We conclude that synthesized production of stable SIgA can be generated by co-purification. This process introduces a simplified means of generating a variety of pathogen-specific SIgA antibodies for research and clinical applications.
authors.affiliation: Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Moldt
authors.affiliation: Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Saye-Francisco
authors.affiliation: Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Niccole
authors.initials: N
authors.lastname: Schultz
authors.affiliation: Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Boston, MA, USA.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Burton
authors.affiliation: Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA. Electronic address: hessell@ohsu.edu.
authors.forename: Ann J
authors.initials: AJ
authors.lastname: Hessell
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000096:biosynthesis* / Q000302:isolation & purification; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D015699:HIV Envelope Protein gp120 / Q000276:immunology; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D007071:Immunoglobulin A, Secretory / Q000096:biosynthesis* / Q000302:isolation & purification; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D057134:Antibodies, Neutralizing; D015699:HIV Envelope Protein gp120; D007071:Immunoglobulin A, Secretory; D011994:Recombinant Proteins; C514793:gp120 protein, Human immunodeficiency virus 1
keywords: Affinity chromatography; Antibody; CHO; Envelope HIV-1 Env; HIV-1; IgA; IgG; J chain; Mucosal IgA; PCs; SC; SHIV; SIgA; Secretory IgA; chinese hamster ovary; dIgA; dimeric IgA; immunolglobulin A; immunolglobulin G; joining chain; mAb; mIgA; monoclonal antibody; monomeric IgA; pIgR; plasma cells; polymeric Ig receptor; recombinant human secretory component; rhSC; secretory IgA; secretory component; simian/human immunodeficiency virus